Diabetic nephropathy (DN) is one of the major complications of diabetes that can lead to end stage renal disease. Key features of DN include increased glomerulosclerosis and mesangial cell widening due to extracellular matrix (ECM) deposition. Although several biochemical pathways and key profibrotic factors, such as transforming growth factor-beta 1 (TGF-) and the ECM protein collagen, have been implicated in the pathogenesis of DN, the subtle molecular and nuclear mechanisms regulating them are unclear. We recently uncovered a novel connection between key gene targets of TGF- in mesangial cells (MCs) and members of a family of small non-coding RNAs called micro-RNAs (miRs). Increasing evidence shows that miRs play important roles in gene regulation since they can suppress the expression of target genes by binding to their mRNAs. Although several targets of miRs have been predicted, much needs to be done to determine their biological and disease relevance. The mechanisms by which miRs are regulated under normal and disease conditions are also not clear. We propose to explore these aspects in the context of DN. We discovered that TGF- downregulates key repressive factors involved in collagen regulation, and that these factors are also targeted and downregulated by two specific miRs expressed in the kidney and MCs. The expressions of these miRs are increased in MCs treated with TGF- and also in the glomeruli of diabetic mice. Furthermore, we observed that these miRs can trigger regulatory and feed forward mechanisms that enhance the expression of collagen. We therefore put forward the novel hypothesis that increased TGF- in DN leads to the aberrant production and actions of key miRs and their target genes which result in enhanced glomerulosclerosis. Specific Aim 1 will examine the transcriptional mechanisms by which TGF- upregulates a master miR and its downstream effector miR in MCs. Specific Aim 2 will evaluate the specific gene target of the downstream miR and how it contributes to TGF- mediated collagen regulation. Specific Aim 3 will test the in vivo functional relevance by evaluating the therapeutic potential of anti-miR oligo-nucleotides in diabetic mice, and by examining the progression of DN in a miR knockout mouse. Our preliminary results have uncovered novel hitherto unexplored mechanisms of action of TGF-. This state-of-the-art study could break new ground and have a major impact in the field of renal research by deciphering the biological functions of these elusive small RNAs in the kidney. They could also pave the way for novel new therapies for diabetic kidney disease. PUBLIC HEALTH RELEVANCE: Diabetes is highly prevalent in the USA and a major healthcare problem. This is further magnified by the fact that it is associated with significantly accelerated and debilitating complications such as diabetic nephropathy. This project proposes to identify novel new mechanisms involved that could lead to the development of sorely needed newer therapies to reduce the morbidity and mortality of diabetic nephropathy.